Figure 1: Nerve injury blocks vesicle trafficking in both severed and intact axons. (See also Figure S1.).

(A) Schematic of injury and vesicle transport live imaging assay in Drosophila wings. Adult fly wings were axotomized, animals were incubated, after which wings were dissected and imaged. Individual clones were labeled using MARCM (green), which allowed for clone-specific expression of markers; severed and intact axons were identified by tracing to cell bodies; unlabeled neurons (gray). (B) Representative time series and kymographs of mCherry-atg8a (arrows) transports in axons of uncut (left column), severed axons 3 hours post axotomy (middle column), and intact neurons 3 hours post axotomy (hpa) (right column). Autophagosomes (arrowheads) were highly motile in uncut axons but exhibited reduced movement in severed and intact axons by hours 3hpa. The individual autophagosome puncta were labeled with yellow lower-case alphabet in the montage images. Scale bar,10 μm. Time is total 3 minutes. (C, D) Quantification of moving autophagosomes over time in (C) severed axons or (D) proximal intact axons. Ordinary one-way ANOVA with Sidak multiple comparisons test. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 10 axons of each, Error bar = S.E.M.). (E) Upper panel: Schematic showing distal wing nerve injury and sites where intact axons were subsequently imaged (site A and B) 3hpa. Lower panel: Quantification of autophagosome movement. Ordinary one-way ANOVA with Sidak multiple comparisons test. (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 10 axons of each, Error bar = S.E.M.).